The Role Of Notch4Signaling Of Endothelial Progenitor Cells In Kawasaki Disease Model With Coronary Artery Lesions

Background:Kawasaki disease, a self-limited systemic vascular inflammatory syndrome characterized by acute rash and fever, predominantly affects infants and children under5years of age. It belongs to autoimmune inflammatory disease and was first reported by the Japanese pediatrician, Tomisaku Kawasaki, in1967. It was regarded as a rare disease at first, but now it has been replaced rheumatic fever as the primary causes of acquired heart disease in children in many countries (it was seen more in Asian countries), and it is also one of the common cause of adult cardiovascular disease in our country. Endothelial cells shedding can lead to vascular intima damage, exposure of smooth muscle layer and collagen, platelet aggregation in the course of Kawasaki disease. These pathological processes in turn increased inflammation, causing destruction of blood vessels structural integrity, and eventually led to coronary artery stenosis or occlusion and even coronary artery aneurysms. EPCs, the precursor cells of endothelial cells, maintain endothelial integrity, promote endothelial regeneration and play an important role in the formation of new blood vessels. When tissue ischemia or vascular injury occur, EPCs will be released to peripheral blood from bone marrow, and differentiation, proliferation, migration and integration to part of endothelial injury will be involved in endothelial repair and micro vascular generation process. Promoting the differentiation and mobilization of EPCs could increase the number of peripheral blood circulating EPCs, which will be the new direction of the treatment for coronary artery lesions. At present, the definition and characteristics of endothelial progenitor cells, the concrete process of mobilization, migration, differentiation, homing and detailed mechanism are not clear or very controversial. Communication between cells in cardiovascular development mediated by Notch4signaling plays an important role in the process of vascular repair. Notch4signaling pathway is the key of maintaining hematopoietic stem cells and EPCs etc. undifferentiated state. Notch4receptors of endothelial progenitor cells, combining with the corresponding ligands, could promote proliferation, differentiation and arteriovenous transformation of endothelial progenitor cells and maintain stem cells undifferentiated state. Notch4signaling plays an critical regulatory role in angiogenesis. So the Notch4signaling has a close relationship with differentiation and mature of endothelial progenitor cells.Given that etiology and pathogenesis of Kawasaki disease are still unclear, the clinical studies about the state of EPCs of the children with Kawasaki disease were limited, the interaction between cells, drug intervention and ethical considerations, we induced coronary artery immune pathological model of Kawasaki disease with LCWE. We studied the number and function changes of EPCs and changes of Notch4and its downstream signal factor, RBP-Jκ, in mRNA and protein levels through bone marrow mononuclear cells culture of Kawasaki disease model with coronary artery lesions.Objective:We induced mice model of Kawasaki disease with LCWE and detected the number and function changes of bone marrow EPC in the different stages of disease progression.we also research the effect of Notch4sigaling on coronary artery lesions on mice Kawasaki disease. Methods:Part1:Detection of the functional changes of mouse bone EPCs in vitro culture1. LCWE (Lactobacillus casei cell wall extract) was prepared with Lactobacillus casei.2. BALB/C strain male mice were treated with a single intraperitoneal injection of LCWE to induce the mice model of Kawasaki disease.3. Bone marrow mononuclear cells were cultured in vitro with EGM-2. Following7days of culture, some related tests were performed.4. Cell Proliferation ability:Cell proliferation activity was detected with CCK kit. The absorbance at a wavelength of450nm was determined. Proliferation activity was calculated with OD.5. Cell Adhesion ability:EPCs were suspended in EGM-2in24-well plates pre-coated with fibronectin and incubated for30minutes. After washing with PBS, the attached cells were counted under the microscope.6. Cell Migration Ability:Cell migration ability was assessed with plug-in Transwell24-well plates. Lower membrane migrated cells were counted under the microscope.7. In Vitro Angiogenesis Ability:EPCs were cultured on ECMatrix glue, Cells were incubated at37℃in a humidified environment with5%carbon dioxide (CO2), and small tubes (vascular)were counted under the microscope at4h,6h,8h,10h,12h after culture.8. Immunofluorescence:(1). Double Fluorescence:Cells were incubated with DiI-acLDL and FITC-UEA-I after7days of cells culture. Nuclear was dyed with DAPI, and the cells were then examined using laser scanning confocal microscope. (2). Cells were incubated with RBP-Jκ and VEGFR-2fluorescence antibody respectively. Nuclear was dyed with DAPI, and the cells were then examined using laser scanning confocal microscope.Part2:Detection of the numbers of mice bone marrow EPCs and Notch (+) EPCs1. The bone marrow mononuclear cells of the mice were separated after intraperitoneal injection of LCWE at3d,7d,14d and28d,and the control group mice were treated at the same time. Cells were incubated with antibodies to CD34-eFluro660, Notch4-PE, Flk-1-FITC, and CD45-Percp-cy5.5. The numbers of EPCs and Notch4(+) EPCs were detected by flow cytometry.2. The cultured EPCs were digested with trypsin and then Cells were treated as shown above and detected by flow cytometry.Part3:Detection of the mRNA levels of NOTCH4, RBP-Jκ, P-Selectin and VCAM-1by fluorescent quantitative PCRWe used relative quantitative Real-time PCR to detect mRNA levels of NOTCH4, RBP-Jκ, P-Selectin, VCAM-1of mice bone marrow EPCs in vitro culture cells of the LCWE-treated group and the corresponding controls at the same time.Part4:Detection of expressions of RBP-Jκ and VEGFR-2proteins in vitro cultured mice bone marrow EPCsWe extracted proteins of mice bone marrow EPCs in vitro culture cells. SDS-PAGE electrophoresis, proteins transferring, blocking, primary antibody incubation, secondary antibody incubation and detection of proteins with chemiluminescence substrate were performed. Finally, we got Western Blot image.Result:Part1:the functional changes and identification of mice bone EPCs in vitro culture 1. Bone marrow EPCs colony forming activity significantly decreased in vitro cultured EPCs in LCWE-treated group compared to controls.2. Bone marrow EPCs proliferation activity significantly decreased in vitro cultured EPCs in LCWE-treated group compared to controls.3. Bone marrow EPCs adhesion ability significantly decreased in vitro cultured EPCs in LCWE-treated group compared to controls.4. Bone marrow EPCs migration ability significantly decreased in vitro cultured EPCs in LCWE-treated group compared to controls.5. In vitro angiogenesis ability of bone marrow EPCs in vitro culture of Kawasaki disease model mice significantly decreased compared to controls.6. Immunofluorescence(1) Double Fluorescence:Double fluorescence staining percentages of bone marrow EPCs in vitro culture of Kawasaki disease model mice were lower than the control group.(2) RBP-Jk positive expression rate were lower in3d,7d,14d group than the control group.(3) VEGFR-2positive expression rate were lower in3d,7d,14d group than the control group.Part2:the numbers of mice bone marrow EPCs and Notch (+) EPCs1. Percentages of bone marrow EPC of LCWE-treated group were lower than controls. Percentages of Notch4(+) EPCs in3d,7d,14d LCWE-treated groups were significantly higher than controls. But the differences between28d LCWE-treated group and the control group have no significant statistical significance.2. Percentages of bone marrow EPC in vitro culture of Kawasaki disease mice model were lower than controls. But the Notch4(+) EPCs levels In LCWE-treated groups were significantly higher than controls.Part3:the mRNA levels of NOTCH4, RBP-Jκ, P-Selectin and VCAM-1in vitro culture bone marrow EPCs1. NOTCH4mRNA level of3d LCWE-treated group was lower than controls.2. RBP-J κ mRNA levels of3d and7d LCWE-treated group were lower than controls.3. P-Selectin mRNA level of3d LCWE-treated group was lower than controls.4. VCAM-1mRNA:Differences between each LCWE-treated group and controls have no significant statistical significancePart4:the protein levels of RBP-Jκ and VRGFR-2in vitro culture bone marrow EPCs1. RBP-Jκ protein of3d LCWE-treated group was lower than controls. Differences between the7d,14d,28d LCWE-treated group and controls have no significant statistical significance.2. VRGFR-2protein:Differences between each LCWE-treated group and controls have no significant statistical significance.Conclusion1. Numbers of EPCs in bone marrow in Kawasaki disease mice model significantly reduced.2. Various functions and bioactivities of bone marrow EPCs in Kawasaki disease mice model were badly impaired.3. Notch4-RBP-JK signaling pathway is involved in damage and repair of Kawasaki disease with coronary artery lesions, and its specific mechanism needs further research.